CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein
Autoři:
Milad Babaei aff001; Yuekun Liu aff001; Shelly M. Wuerzberger-Davis aff002; Ethan Z. McCaslin aff001; Christopher J. DiRusso aff001; Alan T. Yeo aff001; Larisa Kagermazova aff001; Shigeki Miyamoto aff002; Thomas D. Gilmore aff001
Působiště autorů:
Department of Biology, Boston University, Boston, Massachusetts, United States of America
aff001; Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin, United States of America
aff002
Vyšlo v časopise:
PLoS ONE 14(9)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0222588
Souhrn
The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.
Klíčová slova:
DNA transcription – Protein expression – 293T cells – Phosphorylation – DNA cloning – Guide RNA – Cell disruption
Zdroje
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